One glo reagent

For siRNA experiments, HEK293 cells were reverse transfected in 96-well format by incubating 2 pmol lyophilized siRNA in 50 μl OptiMEM with 0.2 μl Lipofectamine RNAiMax (ThermoFisher Scientific) for 30 min and then adding 5000 cells in 50 μl DMEM with 10% FBS, PS/Q. After 48 h, supernatants were removed and cells infected with 1000 TCID₅₀ rgEBOV-luc2 in a volume of 100 μl DMEM with 5% FBS, PS/Q. After an additional 48 h, 100 μl ONE-Glo reagent (Promega) was added to the cells, and reporter activity was measured after 10 min using a Glomax Multi microplate reader. Four biological replicates per siRNA in two independent experiments (two biological replicates per experiment) were obtained on a total of eight 96-well plates. On each plate, there were 8 wells each for negative siRNA (aneg #2), mock-infection, and no siRNA controls, and 4 wells each for the AllStars Cell Death Control siRNA (Qiagen) and the anti-EBOV L siRNA controls. For inhibitor experiments, HEK293 (EBOV, NDV, RABV) or VeroE6 (EBOV, IAV) cells were infected with GFP-expressing viruses at an MOI of 0.1 (EBOV, RABV, IAV) or 0.05 (NDV). Supernatants were harvested 48 h post infection, and titers were determined by TCID₅₀ analysis. To assess the effects on cell viability, a CellTiter-GLO assay (Promega) was performed in parallel following the manufacturer’s instructions.

All reagents were of analytical grade. Dulbecco’s modified Eagle medium (DMEM), RPMI 1640 medium and fetal bovine serum (FBS) were from GIBCO Life Technologies (Grand Island, NY, USA). Penicillin, streptomycin, Triton-X100, BriJ, sodium orthovanadate, protease inhibitor cocktail, and anti-α-tubulin were from Sigma-Aldrich (St. Louis, MO, USA). Bradford reagent was from Bio-Rad Laboratories (Milan, Italy). Trizol, MMLV reverse transcriptase and CellTracker Red CMTPX Dye were from Invitrogen (Carlsbad, CA, USA). PVP-free polycarbonate filters were obtained from Costar (Cambridge, MA, USA). Diff-Quik reagent was obtained from Dade-Behring (Deer eld, IL, USA). ONE-Glo™ Reagent and DNAse were from Promega (Milan, Italy). Recombinant FGF2 was purchased from Tecnogen (Caserta, Italy). Anti-FGFR1, anti-ERK_1/2, anti-phospho-ERK_1/2 (Thr202/Tyr204), anti-phospho-AKT (Ser473), anti-cleaved-PARP, anti-cleaved-caspase-3, and anti-β-catenin were from Cell Signaling Technologies (Danver, MA, USA). Anti-phospho-FGFR1 (Tyr766), anti-phospho-FRS2 (Tyr196), anti-FGFR3, and anti-GAPDH were from Santa Cruz (Santa Cruz, CA, USA). Anti-phospho-FGFR3 (Tyr724) was from ABCAM (Cambridge, UK). Matrigel was from Cultrex BME (Gaithersburd, MD, USA). Floseal hemostatic matrix was from Baxter (Deerfield, IL, USA).

HEK293T-Rex cells incorporating either the ERSE-FLuc or XBP1s-RLuc reporters were collected by trypsinization and resuspended at a density of 500,000 cells per mL. The assay was started by dispensing 5 μL of cell suspension into each well of white, solid-bottom 1536-well plates using a flying reagent dispenser (FRD) and placed into an online incubator for 3 hr. Cells were then treated with 34 nL/well of either test compounds to give final concentrations of 6.8 μM, DMSO (low control, final concentration 0.68%, 0% activation) or 37 μM of Delta-7 thapsigargin (high control, final concentration 500 nM, 100% activation). Plates were incubated for 18 hr at 37°C, removed from the incubator and equilibrated to room temperature for 10 min. Luciferase activity was detected by addition of 5 μL of ONE-Glo reagent (Promega) to each well. After a 10 min incubation time, light emission was measured with the ViewLux reader (PerkinElmer). The percent activation of each test compound was calculated as follows: % Activation = 100*(Test Compound- Median Low Control) / (Median High Control – Median Low Control).

Codon-optimized full-length spike (S) protein of the original Wuhan-Hu-1 isolate with D614G mutation (D614G) was cloned into a pCAGGS vector. This codon-optimized D614G vector was used as a template for site-directed mutagenesis to incorporate the S variants, listed in Table 1. To make SARS-CoV-2 full-length S-pseudotyped recombinant vesicular stomatitis virus ΔG (VSVΔG)-firefly luciferase virus, BHK-21/WI-2 cells (Kerafast) were transfected with the S expression plasmid and subsequently infected with VSVΔG-firefly luciferase as previously described (11 (link)). For the neutralization assay, serially diluted serum samples were mixed with pseudovirus and incubated at 37°C for 45 min. The virus-serum mix was subsequently used to infect A549-hACE2-TMPRSS2 cells (12 (link)) for 18 h at 37°C before addition of ONE-Glo reagent (Promega) for measurement of the luciferase signal by relative luminescence units (RLUs). The percentage of neutralization was calculated based on the RLUs of the virus-only control and subsequently analyzed using the four-parameter logistic curve in Prism v.8 (GraphPad Software, Inc.).

Lentiviral particles containing luciferase reporter construct driven by NF-κB (System Biosciences) were obtained and transfected into BT474 and BT474 PTEN⁻ LTT cells. Two hours following incubation with SF, TNF-α concentration in media was brought to 50 ng/ml. After 6 hr Luciferase activity was measured according to manufacturer’s instructions with oneGlo reagent (promega) on Synergy 2 plate reader (BioTek).

In vitro translation assays were performed with the Retic Lysate IVT Kit (Invitrogen-AM1200, Villebon Sur Yvette, France) following the manufacturer’s instructions and using the F-Luc mRNA (12.5 ng/µL) as a reporter. The translation efficiency of this transcript was analyzed in the presence of either P. lividus eIF4B or R-Luc mRNAs, both at a final concentration of 12.5 ng/µL. Incubations were performed at 30 °C in an Eppendorf ThermoMixer with agitation at 300 rpm. For each time point, 10 µL of the reaction assay was mixed with 50 µL of ONE-Glo reagent (Promega-E6110, Charbonnières-les-Bains, France), and the sample luminescence was measured for 10 s on a 96-well microplate using a Tristar luminometer (Berthold).